Design, synthesis and evaluation of quinazolinone analogues as monoamine oxidase inhibitors

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Design, synthesis and evaluation of

quinazolinone analogues as monoamine oxidase

inhibitors

MA Qhobosheane

orcid.org 0000-0003-3005-0028

Dissertation submitted in partial fulfilment of the requirements

for the degree Magister of Science in Pharmaceutical

Chemistry at the North-West University

Promoter:

Prof LJ Legoabe

Co-promoter:

Prof JP Petzer

Assistant promoter:

Prof A Petzer

Graduation May 2018

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This work is based on the research supported by National Research Foundation of South Africa (Grant specific unique reference numbers (UID) 96135). The Grant holders acknowledge that opinions, findings and conclusions or recommendations expressed in any publication generated by the NRF supported research are that of the authors, and that the NRF accepts no liability whatsoever in this regard.

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PREFACE

This dissertation is submitted in an article format in accordance with the General Academic Rules (A.13.7.3) of the North-West University. This dissertation includes two articles which were compiled for submission to Bioorganic & Medicinal Chemistry. The author guidelines have been included (Appendix C). All scientific research (synthesis, biology and documentation of the dissertation and articles) for the purpose of this dissertation was conducted by Miss M.A. Qhobosheane at the North-West University, Potchefstroom campus.

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ACKNOWLEDGEMENTS

First and foremost I would like to thank the Almighty God for granting me the strength and courage to finish this dissertation.

I would like to express my sincere appreciation to the following people for all the support they showed me throughout the course of my study:

 My supervisor Prof L.J. Legoabe for your constant encouragement, guidance and patience. Words cannot describe how grateful I am to have had you as my mentor. It was a great honour to be in your group.

 My co-supervisor Prof J.P. Petzer for your expertise.

 My assistant supervisor Prof A. Petzer for your advice and assistance with the biological studies.

 All Pharmaceutical Chemistry personnel for your co-operation and creating a friendly and enabling environment.

 My mother Maagatha, my brother Nkeeane and my sister Mabohlokoa Qhobosheane for your constant love, support and encouragement.

 My best friend Teboho Khofu for your love and support in difficult times during my study.  All my friends and family for your love and support.

 Reformed Church Potchefstroom, die Bult bible study group for your spiritual support and constant encouragement.

I would also like to thank the following institutions for their assistance during this study:

 The North-West University for granting me an opportunity to study at this institution and for the financial support.

 Dr J. Jordaan and Mr A. Joubert of the SASOL Centre for Chemistry for your help with NMR and MS analyses.

 Prof Jan du Preez for your help with the HPLC analyses.

“No discipline seems pleasant at the time, but painful. Later on, however, it produces a harvest of righteousness and peace for those who have been trained by it”

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ABSTRACT

Parkinson’s disease (PD) is the second most common neurodegenerative disease after Alzheimer’s disease, and it is estimated to affect approximately 1% of the population over the age of 65. PD is characterised by non-motor and motor symptoms such as resting tremor, bradykinesia and muscle rigidity, which are a result of neuronal dopamine deficiency due to the progressive loss of the dopaminergic pathway that leads from the substantia nigra pars compacta (SNpc) to the striatum. Non-motor symptoms of PD include sleep disturbances, depression and anxiety.

There is presently no cure for PD, and the present treatment can neither reverse nor stop the disease progression. However, PD can be treated symptomatically with a variety of therapies which include L-dopa, dopamine agonists, aromatic L-amino acid decarboxylase (AADC) inhibitors, catechol-O-methyltransferase (COMT) inhibitors and monoamine oxidase (MAO) B inhibitors. L-dopa has been the mainstay of PD treatment for over 30 years, and it remains the most effective treatment to date. However, L-dopa should be combined with a peripheral AADC inhibitor to ensure its neuronal bioavailability and to avoid its peripheral side-effects. MAO-B inhibitors have also been found effective in PD treatment because they enhance brain dopamine levels in PD, and thus alleviate the symptoms.

The MAO-A and MAO-B enzymes are mitochondrial outer membrane-bound flavoproteins that catalyse the oxidative deamination of monoamine neurotransmitters dopamine, norepinephrine and epinephrine. The MAOs are differently distributed in the body, with MAO-A dominating in the intestines, heart and placenta, while MAO-B dominates in the brain, glial cells in the brain and liver. Oxidation of dopamine by MAO generates hydrogen peroxide and aldehyde derivatives, by-products which are potentially neurotoxic. MAO-B inhibitors increase brain dopamine levels and also reduce levels of hydrogen peroxide and aldehyde derivatives in the brain, and therefore are neuroprotective in this respect. MAO-A inhibitors are used clinically in treatment of depression, while MAO-B inhibitors are used as therapy for PD. Selective and reversible MAO inhibitors are more clinically acceptable because they do not cause the side-effects that are associated with irreversible and non-selective MAO inhibition.

The aim of the present study was to explore 4(3H)-quinazolinone as a scaffold for design of potent and selective MAO-B inhibitors.

The MAO inhibitory potential of quinazolinones has been illustrated in several studies. A study conducted by Bahadur (1982) revealed that quinazolinones can inhibit MAO activity by as much as 80%. In their study, Bahadur (1982) discovered that the increase or decrease in MAO inhibitory activity of quinazolinones depends on the type of substituent, as well as the position at which it is attached. This is in agreement with similar studies carried out by Rastogi et al. (1972)

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and Lata et al. (1982). A number of studies have been conducted to evaluate quinazolinones as potential MAO inhibitors, but none have been done to study the structure-activity relationships (SARs) with respect to thiobenzyl and benzyloxy substitution. This study expanded on the SARs of MAO inhibition by quinazolinone derivatives to enable the design of novel potent MAO inhibitors of this chemical class. Particular attention was given to the benzyloxy and thiobenzyl derivatives of 4(3H)-quinazolinone.

Chemistry: Two series of compounds were synthesised and evaluated as potential MAO inhibitors. The thioether (14 compounds), C6 mono- (12 compounds) and N3/C6 disubstituted (9 compounds) derivatives of 4(3H)-quinazolinone were synthesised using standard chemical procedures. The reactants were suspended in either ethanol or N,N-dimethylformamide (DMF) in the presence of a base. The products were precipitated with ice-cold water and were subsequently dried or recrystallised from appropriate solvents. The structures and purities were confirmed by NMR, MS and HPLC.

MAO inhibition studies: To determine the 50% inhibitory concentration (IC50) values and selectivity index (SI), a fluorometric assay was carried out employing recombinant human MAO-A and MMAO-AO-B as enzyme sources, and kynuramine as substrate. The first series consisted of 14 compounds, 12 of which exhibited good MAO-B inhibition properties, with IC50 values in the micromolar to sub-micromolar range. The most potent compound in this series (3k) exhibited an

IC50 value of 0.142 µM. Interesting trends were observed through the SAR analyses of the compounds in this series. For example, meta-halogen substitution of the thioether derivatives dramatically increased the inhibitor potencies. A number of derivatives (5 of 21) in the second series showed selective inhibition of MAO-B. The disubstituted compounds 2b and 2h are

notable as the most potent inhibitors in this series with IC50 values of 0.685 µM and 0.847 µM, respectively. However, meaningful SARs for MAO inhibition could not be derived because most compounds in this series did not inhibit the MAOs.

The 4(3H)-quinazolinone derivatives were successfully synthesised in this study, and it may be concluded that they are potent and selective MAO-B inhibitors, thus promising leads for the future design of PD therapies.

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2.3.1.3 IC50 determination ... 54 2.4 Animal models of PD ... 55 2.4.1 MPTP ... 55 2.4.1.1 General background ... 55 2.4.1.2 Mechanism of action ... 55 2.4.2 6-OHDA ... 56 2.4.3 Rotenone ... 57 2.4.4 Paraquat ... 58 2.4.5 Gene-based models ... 58 2.5 Quinazolinones ... 59 2.5.1 General background ... 59 2.5.2 Biological activities ... 60 2.5.3 Synthetic methods ... 61 2.6 Conclusion ... 62 REFERENCES ... 63

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ix CHAPTER 3: ARTICLE 1 ... 71 REFERENCES ... 86 APPENDIX A: SPECTRA ... 88 CHAPTER 4: ARTICLE 2 ... 117 REFERENCES ... 135 APPENDIX B: SPECTRA ... 138 CHAPTER 5 CONCLUSION ... 181 REFERENCES ... 184

APPENDIX C: AUTHOR GUIDELINES ... 186

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LIST OF TABLES

Table 1.1: Proposed compounds to be synthesised in this study ... 12 Table 2.1: Distribution of MAO-A and MAO-B in man and in the brains of selected

species ... 38 Table 2.2: Substrate specificities of MAO in the cerebral cortex. ... 39

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LIST OF FIGURES

Figure 1.1: Some common MAO inhibitors used in the treatment of depression and

Parkinson’s disease. ... 8

Figure 1.2: Quinazolinone derivatives found to actively inhibit MAO in previous studies (Bahadur, 1983; Khattab et al., 2015). ... 10

Figure 2.1: Neuropathology of PD (Dauer & Przedborski, 2003). ... 20

Figure 2.2: Structures of dopamine and L-dopa. ... 22

Figure 2.3 Structures of bromocriptine, ropinirole, pramipexole and pergolide. ... 23

Figure 2.4: Structure of apomorphine. ... 24

Figure 2.5: Structures of carbidopa and benserazide ... 24

Figure 2.6: Structures of tolcapone and entacapone ... 25

Figure 2.7: Structures of selegiline and rasagiline ... 26

Figure 2.8: Structures of trihexyphenidyl, diphenhydramine, and benztropine. ... 27

Figure 2.9: Structure of istradefylline (KW-6002) ... 28

Figure 2.10: Structure of amantadine ... 28

Figure 2.11: Structure of lazabemide. ... 29

Figure 2.12: Structures of coenzyme Q10 and creatine ... 30

Figure 2.13: Structures of minocycline, TCH346 and Cep-1347 ... 32

Figure 2.14: Genetic mutations and pathogenesis of PD (Vila & Przedborski, 2004). ... 35

Figure 2.15: Structure of pargyline ... 44

Figure 2.16: Structure of ladostigil ... 45

Figure 2.17: Structure of isatin ... 45

Figure 2.18: Structure of (E)-8-(chlorostyryl)caffeine ... 46

Figure 2.19: Structure of 1,4-diphenylbutene ... 46

Figure 2.20: Structure of trans,trans-farnesol ... 46

Figure 2.21: Structure of safinamide ... 47

Figure 2.22: Structure of clorgiline ... 47

Figure 2.23: Structures of tranylcypromine and phenelzine ... 48

Figure 2.24: Structures of moclobemide and brofaromine ... 48

Figure 2.25: Structure of iproniazid ... 49 Figure 2.26: Structure of human MAO-B. The covalent flavin moiety is shown in a ball

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substrate domain is in red and the membrane binding domain is in green

(Edmondson et al., 2007). ... 50

Figure 2.27: Ribbon diagram of the human MAO A structure. The covalent flavin moiety is shown in a ball and stick model in yellow. The flavin binding domain is in blue, the substrate domain is in red and the membrane binding domain is in green (Edmondson et al., 2007)... 51

Figure 2.28: Conversion of kynuramine to 4-hydroxyquinoline by MAO (Yan et al., 2004). ... 52

Figure 2.29: The conversion of MPTP to MPP+_{... 56}

Figure 2.30: Structure of 6-OHDA ... 57

Figure 2.31: Structure of rotenone ... 58

Figure 2.32: Structure of paraquat ... 58

Figure 2.33: Schematic representation of the site of action of pharmacological agents or genetic manipulations resulting in nigrostriatal degeneration and striatal DA depletion. (Betarbet et al., 2002). ... 59

Figure 2.34: Isomers of quinazolinones ... 60

Figure 2.35: Structure of 2-cyano-3,4-dihydro-4-oxoquinazoline ... 61

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List of abbreviations

5-HIAA 5-Hydroxyindole acetic acid 5-HT Serotonin

6-OHDA 6-Hydroxydopamine

AADC Aromatic L-amino acid decarboxylase ACh Acetylcholine

ADH Alcohol dehydrogenase ALDH Aldehyde dehydrogenase

APCI Atmospheric pressure chemical ionisation ATP Adenosine triphosphate

BBB Blood-brain barrier

BDNF Brain-derived neurotrophic factor BH4 Tetrahydrobiopterin

ChE Cholinesterase

CNS Central nervous system

COMT Catechol-O-methyl transferase COX Cyclooxygenase

CSC (E)-8-(Chlorostyryl)caffeine CSF Cerebrospinal fluid

DA Dopamine

DARPP Dopamine- and cAMP- regulated phosphoprotein DAT Dopamine transporter

DATATOP Tocopherol and deprenyl antioxidative therapy of parkinsonism DMD Dimethylformamide

DMSO Dimethyl sulfoxide DNA Deoxyribonucleic acid

DOPAC 3,4-Dihydroxyphenylacetic acid DOPAL 3,4-Dihydroxyphenylacetaldehyde DOPET 3,4-Dihydroxyphenylethanol FAD Flavin adenine dinucleotide GABA γ-Aminobutyric acid

GAPDH Glyceraldehyde-3-phospjate dehydrogenase GBA Glucocerebrocidase

GDNF Glial-derived neurotrophic factor GI Gastrointestinal

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iii GPO Glutathione peroxidase

GSH Glutathione

GTP Guanosine triphosphate GTPCH GTP cyclohydrolate H2O2 Hydrogen peroxide

HPLC High performance liquid chromatography HRMS High resolution mass spectra

IC50 50% inhibitory concentration

IL Interleukin

JEV Japanese encephalitis virus JNK c-Jun N-terminal kinase

LB Lewi body

L-dopa Levodopa

LRRK-2 Leucine rich repeat kinase-2 MAO Monoamine oxidase

MAPK Mitogen-activated protein kinase MHz Megahertz mp Melting point MPDP+ _{1-Methyl-4-phenyl-2,3-dihydropyridine} MPP+ _{1-Methyl-4-phenylpyridinium} MPPP 1-Methyl-4-phenyl-4-propionoxipiperidine MPTP 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine mRNA Messenger ribonucleic acid

NADH Nicotinamide adenine dinucleotide

NADPH Nicotinamide adenine dinucleotide phosphate NGF Nerve growth factor

NMDA N-Methyl-D-aspartate

NMR Nuclear magnetic resonance

NO Nitric oxide

NOS Nitric oxide synthase

NSAIDs Non-steroidal anti-inflammatory drugs ONOO- _{Peroxynitrite}

PD Parkinson’s disease

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iv PNS Peripheral nervous system

PTEN Phosphatase and tensin homologue RNA Ribonucleic acid

ROS Reactive oxygen species SARs Structure-activity relationships SD Standard deviation

SI Selectivity index

SNpc Substantia nigra pars compacta SOD Superoxide dismutase

SSRIs Selective serotonin reuptake inhibitors t1/2 Half-life

TB Tuberculosis

TCAs Tricyclic antidepressants TH Tyrosine hydroxylase TLC Thin layer chromatography TNF Tumour necrosis factor

UCH-L1 Ubiquitin carboxyl terminal hydrolase L1 VMAT2 Vesicular monoamine transporter 2 VTA Ventral tegmental

Kinetics EI Enzyme-inhibitor complex ES Enzyme-substrate complex I Inhibitor Ki Inhibition constant Km Michaelis constant [S] Substrate concentration Vi Initial velocity

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CHAPTER 1 INTRODUCTION

1.1 Introduction

James Parkinson initially described Parkinson’s disease (PD) as involuntary tremulous motion, with decreased muscle power at rest (Parkinson, 2002), which diminishes during voluntary activity (Rang et al., 2007) and is accompanied by slowness of movement and impairment of postural balance with a tendency to bent the trunk forward and to pass from a walking to a running pace. The senses and intellect remain unharmed in PD (Parkinson, 2002). PD is a progressive disorder whose symptoms worsen with time (Roach & Scherer, 2004), and the symptoms usually begin on one side of the body and gradually spread to the other side (Fahn, 2003).

PD is the second most common age-related neurodegenerative disorder after Alzheimer’s disease, and it affects approximately 1% of the population over the age of 65 (Gibrat et al., 2009). According to Rang et al. (2007) this disease has no known underlying cause, but is the result of a deficiency of dopamine (DA) and an excess of acetylcholine (Ach) in the central nervous system (CNS) (Roach & Scherer, 2004). Today, PD is characterised by resting tremor which decreases with voluntary movement, increased resistance to passive movement of the limbs (rigidity), slowness of movement (bradykinesia), reduction in movement amplitude (hypokinesia) and absence of normal unconscious movements such as arm swing in walking (akinesia) (Dauer & Przedborski, 2003).

Pathologically, PD is characterised by the progressive loss of dopaminergic neurons projecting from the substantia nigra pars compacta (SNpc) to the striatal motor loci (Gibrat et al., 2009). Cell loss in the locus ceruleus, dorsal nuclei of the vagus, raphe nuclei, nucleus basalis of the Meynert and some other catecholaminergic brain stem structures, including the ventral tegmental area, also exist (Lees et al., 2009). According to Vila and Przedborski (2004), the presence of intraneuronal inclusions called Lewi bodies (LBs) is one of the hallmarks of PD. Susceptible genes such as α-synuclein, leucin rich repeat kinase 2 (LRRK-2) and glucocerebrocidase (GBA) have shown that genetic predisposition is another important causal factor (Lees et al., 2009).

Age is the major risk factor for PD (Lees et al., 2009). The mean age of onset is 55 years (Dauer & Przedborski, 2003), although 10% of people with the disease are younger than 45 years of age (Lees et al., 2009). The incidence of PD increases significantly with age, from 200/100 000 overall to 200/10 000 at age 70, and it seems to decrease in the ninth decade of life (Dauer & Przedborski, 2003; Lees et al., 2009). There is no apparent genetic cause in

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about 95% of PD cases, but the disease is inherited in the remaining cases (Dauer & Przedborski, 2003).

1.2 Monoamine oxidase

Monoamine oxidase (MAO) is an enzyme that is found in all tissues and almost all cells of the body, bound to the outer mitochondrial membrane. Its active site contains flavin adenine dinucleotide (FAD), which is bound to the cysteine of a –Ser-Gly-Gly-Cys-Tyr– sequence (Prisinzano, 2006). MAO catalyses the oxidative deamination of catecholamines such as norepinephrine, epinephrine and DA, as well as the monoamines serotonin and histamine, to the corresponding aldehyde and free amine, with the generation of hydrogen peroxide (H2O2) (Youdim et al., 2006).

MAO is not a single enzyme, but it exists in two forms, MAO-A and MAO-B (Youdim et al., 2006). These isoforms are differently distributed in the body. MAO-B is found predominantly in the brain and platelets, whereas MAO-A is found predominantly in the intestinal tract. MAO-A and MAO-B differ in their substrate preferences, immunological properties, molecular weight and anatomical locations, and they are inhibited by different inhibitors (Victor & Waters, 2003).

During development, MAO-A appears before MAO-B, with the level of MAO-B increasing greatly in the brain after birth (Youdim et al., 2006). The distribution of the two MAO isoforms also differs in the human brain; the highest MAO-A concentrations are in the catecholaminergic neurons of the locus ceruleus, whereas the highest MAO-B concentrations are in the serotonergic and histaminergic neurons of the raphe and posterior hypothalamus (Foley et al., 2000; Youdim et al., 2006). Foley et al. (2000) notes that there are high concentrations of both forms of the MAO enzyme in the human basal ganglia. Youdim et al. (2006) indicates that MAO in peripheral tissues oxidise amines and prevent their entry into the systemic circulation, and thus serve as protective barriers. For example, MAO-B in the microvessels of the blood-brain barrier (BBB) metabolises amines thereby preventing their entry into the CNS (Youdim et al., 2006; Legoabe et al., 2012a). Another example of the protective effect of MAO is found with the “cheese reaction”. Tyramine, an indirectly acting sympathomimetic amine which is present in fermented foods, is metabolised by intestinal MAO-A. This reduces the amount of tyramine that enters the systemic circulation and prevents the tyramine induced release of norepinephrine from peripheral neurons known as the “cheese reaction” (Legoabe et al., 2012a). In addition, intraneuronal MAO-A and MAO-B in the CNS and peripheral nervous system (PNS) protect neurons from

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exogenous amines, terminate the actions of amine neurotransmitters, and regulate the contents of intracellular amine stores (Youdim et al., 2006).

Oxidant stress may occur via the formation of H2O2 and oxygen derived free radicals in the MAO catalytic cycle (Fahn & Cohen, 1992). Because the substantia nigra is rich in DA, which can undergo both enzymatic oxidation via MAO and non-enzymatic autoxidation, H2O2 and oxyradicals are generated in the midbrain nucleus (Fahn & Cohen, 1992). Oxidant stress may cause loss of monoaminergic neurons in patients with PD. Neurotoxins that selectively destroy dopaminergic neurons in the nigra, such as 6-hydroxydopamine

(6-OHDA)

and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) appear to act by

generating oxidant stress (Fahn & Cohen, 1992).

MAO inhibition interrupts the metabolism of catecholamines leading to an increase in endogenous and exogenous substrates as well as trace amines (tyramine, tryptamine etc.). MAO inhibition thus increases the levels of biogenic amine neurotransmitters (Volz & Gleiter, 1998). MAO inhibitors are primarily used for the treatment of depression and neurological disorders such as PD (Volz & Gleiter, 1998). These inhibitors belong to the earliest drugs used in PD, and they have been used for many years alone or in combination with L-dopa, the metabolic precursor of DA (Riederer & Laux, 2011). However, earlier use of non-selective irreversible MAO inhibitors was terminated due to their ability to cause fatal drug-food interactions (e.g. the “cheese reaction”) (Kumar et al., 2016; Schatzberg & Nemeroff, 2017). When MAO-A is inhibited in the PNS, tyramine enters the blood stream (Robakis & Fahn, 2015) and acts as a false neurotransmitter at nerve terminals, triggering the release of norepinephrine which results in hypertensive crisis.

It is worth noting that selective irreversible MAO inhibitors lose their selectivity at high concentrations (Foley et al., 2000). For example, when given at high doses, selegiline, an irreversible selective MAO-B inhibitor, also inhibits MAO-A and potentiates the sympathomimetic action of tyramine (Youdim & Weinstock, 2004). The development of selective MAO inhibitors which are reversible in nature emerged in recent years (Schatzberg & Nemeroff, 2017). At normal doses, MAO-B inhibitors do not act on tyramine metabolism in the gut, while possessing the ability to increase striatal neuronal responses to DA. Therefore MAO-B inhibitors are useful in the treatment of PD (Robakis & Fahn, 2015). Selegiline was the first synthesised selective MAO-B inhibitor, and because of its inability to cause the cheese reaction (at normal doses), it has served as the benchmark against which new MAO-B inhibitors are measured (Foley et al., 2000).

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There is evidence that if MAO-B inhibitors are used in the very early stages of PD, they may delay the need to start L-dopa therapy (Youdim & Weinstock, 2004), and delay the onset of more severe disability (Rossiter et al., 2012). Based on the clinical utility of MAO inhibitors, the discovery of new classes of MAO inhibitors is merited. The current study is focused on exploring the quinazolinone scaffold for the design and synthesis of potent reversible MAO inhibitors.

Figure 1.1: Some common MAO inhibitors used in the treatment of depression and Parkinson’s disease.

1.3 Rationale

Quinazolinone is a class of fused heterocycles that are found in approximately 150 naturally occurring alkaloids isolated from a number of families of the plant kingdom. Quinazolinones are also found in microorganisms and animals (Arora et al., 2011; Rajput & Mishra, 2012; Banu et al., 2015). Due to their diverse range of biological properties, synthetic methods have been explored to develop quinazoline and quinazolinone derivatives (Khan et al., 2016). The MAO inhibitory profiles of quinazolinones have previously been reported by various researchers (Lata et al., 1982; Gökhan-Kelekçi et al., 2009; Khattab et al., 2015).

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A study carried out by Khattab et al. (2015) revealed that selected amino acid derivatives of quinazolinones (compounds 1, 2 and 3) inhibit MAO competitively, with the majority of tested

compounds exhibiting selectivity for MAO-A over MAO-B. Compounds with shorter linkers between the hydrophobic head and the terminal functional group displayed better inhibition activities. This observation was attributed to differing orientations of the inhibitors in the binding side and the formation of hydrogen bonds with the MAO-A backbone (Khattab et al., 2015). A study conducted by Bahadur (1983) revealed that MAO inhibition potency increases when quinazolinones are substituted with a phenyl at position 2 and an optimum substituent at position 6 and/or position 8. The most potent inhibitors (compounds 4 and 5)

were substituted with a halogen on C6 and these compounds inhibited MAO to a level of more than 80%. This is in agreement with results reported by Rastogi et al. (1972), where the introduction of a halogen substituent on position 6 of the quinazolinone nucleus enhanced the inhibitor potency.

Lata et al. (1982)synthesised and screened 16 quinazolinone derivatives as MAO inhibitors and discovered that their potencies are dependent on the position of attachment of the substituent and on the type of substituent attached. In addition, Srivastava et al. (1980) reported that the potency of MAO inhibition increases when quinazolinones are substituted with halogens on the C6 and C8 positions of the quinazolinone backbone. In summary, the above discussions show that attachment of suitable substituents on the appropriate positions of the quinazolinone moiety is a prerequisite for the development of potent MAO inhibitors of this class.

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Figure 1.2: Quinazolinone derivatives found to actively inhibit MAO in previous studies (Bahadur, 1983; Khattab et al., 2015).

Based on these discussions, this study will examine the MAO inhibitory properties of a series of 4(3H)-quinazolinone derivatives. A total of 35 compounds will be synthesised, with the first series comprising of fourteen different thioether derivatives (3a-m) of 4(3H)-quinazolinone,

and the second series consisting of twelve C6 mono- (1a-l) and nine N3/C6 di-substituted

(2a-i) derivatives of 4(3H)-quinazolinone. 1.4 Hypothesis of this study

As discussed above, the MAO inhibitory activity of the quinazolinone scaffold has been reported in a number of studies. However, the effect of benzyloxy and thiobenzyl substitution of the quinazolinone moiety has not yet been explored. Based on the observation that benzyloxy substitution enhances the MAO-B inhibition potency and selectivity of heterocyclic compounds (Legoabe et al., 2012a; Legoabe et al., 2012b), we predict that benzyloxy substituted 4(3H)-quinazolinones will exhibit similar properties. A previous study carried out on a structurally similar bicyclic scaffold, coumarin, reported that thiobenzyl substitution of the coumarin backbone markedly increases affinity and selectivity for the MAO-B isoenzyme (Catto et al., 2006). We envisage that a similar trend will be observed in thioether-containing 4(3H)-quinazolinone derivatives. It is also hypothesised that further substitution of the phenyl

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ring with halogen (F, Cl, Br, I) and alkyl (CN, CF3) substituents will significantly enhance the inhibition potency of the quinazolinone derivatives.

1.5 Objectives of this study

The main aim of this study is to explore 4(3H)-quinazolinone as a scaffold for design of potent and selective MAO-B inhibitors.

The primary objectives of this study are;

 To design, synthesise and characterise novel quinazolinone derivatives.

 To evaluate the synthesised compounds as recombinant human MAO-A and MAO-B inhibitors by determination of the IC50 values.

 To determine the reversibility of inhibition by the selected active compounds. The recovery of the enzymatic activity after dialysis of enzyme-inhibitor complexes will be evaluated.

 To determine if a selected inhibitor’s mode of inhibition is competitive or non-competitive.  To determine the structure-activity relationships of the synthesised compounds as

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Table 1.1: Proposed compounds to be synthesised in this study

3a 3b _3c

3d 3e 3f

3g 3h 3i

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13 3m 3n 1a 1b 1c 1d 1e 1f 1g 1h 1i 1j 1k 1l 2a

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14

2b 2c _2d

2e

2f 2g

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CHAPTER 2 LITERATURE OVERVIEW

2.1 Parkinson’s disease 2.1.1 General background

Parkinson’s disease (PD) is a slowly progressive CNS disorder characterised by involuntary contraction of skeletal muscles at rest, with a tendency to bend forward and to pass from a walking to a running pace (Parkinson, 2002). The major clinical signs and symptoms for diagnosis of PD are dyskinesia (slowness and poverty of movement), muscular rigidity, resting tremor (which subsides with voluntary movement) and impaired postural movement, leading to tendency to fall forward or backwards when the centre of gravity is displaced (Bruton et al., 2008).

The earliest physical signs of PD occur gradually and may be unnoticed for a long time, and include slight dragging of one foot, fatigue and stiffness, stooped posture, flexion of one arm with lack of swing and poor quality of speech (Lees et al., 2009). As time and disease progress, difficulties increase. For example a change in a patient’s writing occurs, with a tendency to slope usually in an upward direction, and then get smaller and more cramped after a line or two (Parkinson, 2002; Lees et al., 2009). Other symptoms include early loss of smell and sleep disturbances (Lees et al., 2009). Lees et al. (2009) indicates that in the late stages of PD, the face becomes mask-like and open mouthed with a wide-eyed, unblinking stare. All dextrous movements become increasingly difficult and a patient may need help in getting out of bed, bathing, dressing and eating (Lees et al., 2009).

2.1.1.1 Neurochemical and neuropathological features

The pathological hallmark of PD is loss of pigmented dopaminergic neurons in the SNpc that provide dopaminergic innervation to the striatum (caudate and putamen) (Bruton et al., 2008) and the presence of LBs (Dauer & Przedborski, 2003). LBs are intracytoplasmic inclusions that are composed of a variety of proteins such as α-synuclein, parkin, ubiquitin and neurofilaments. They can be found in every affected brain region (Przedborski, 2005), and they are a result of defective response to oxidative neuronal injury (Tugwell, 2008). According to Uhl et al. (1994), the pattern of SNpc cell loss coincides with the level of DA transporter (DAT) messenger ribonucleic acid (mRNA) expression, and it is consistent with the finding that DA depletion is most pronounced in the dorsolateral putamen.

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Figure 2.1: Neuropathology of PD (Dauer & Przedborski, 2003).

The cell bodies of the mesolimbic dopaminergic neurons that reside adjacent to the SNpc in the ventral tegmental (VTA) area are less affected in PD (Dauer & Przedborski, 2003). Research indicates that there is less depletion of DA in the caudate, the main site of projection of these neurons (Dauer & Przedborski, 2003). Cell loss in PD is concentrated in the ventrolateral and caudal portions of SNpc, and the degree of terminal loss in the striatum is more pronounced than the extent of SNpc dopaminergic neuron loss, suggesting that the striatal dopaminergic nerve terminals are the major targets for the degenerative process (Dauer & Przedborski, 2003).

2.1.1.2 Aetiology Environmental factors

Several studies have identified that some environmental influences play an important role in the cause of PD (Warner & Schapira, 2003). Rural living is associated with the agricultural industry, and as a result it has been identified as one of the factors that increase the risk of developing PD. For example, paraquat is structurally similar to the N-methyl-4-phenyl-2,3-dihydropyridinium ion (MPP+_{), the active metabolite of MPTP, and it has been used as a} herbicide (Dauer & Przedborski, 2003). High PD incidence has also been shown in people who drink well water (Tugwell, 2008).

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Genetic factors

Genetic mutation is one of the causes of PD aetiology. A greater percentage of PD cases is not inherited, but it has been estimated that a parent with PD increases the child’s lifetime risk of developing the disease from 2% to 6% (Tugwell, 2008). Mutations in the SNpc gene are rare and highly penetrant, and they cause early onset autosomal dominant inherited forms of PD (Polito et al., 2016). On the other hand, mutations in the LRRK-2 gene are rare and have incomplete and age-dependant penetrance. These mutations cause late onset autosomal dominant inherited forms of PD (Polito et al., 2016).

2.1.1.3 Pathogenesis

The symptoms of PD result from the degeneration of the dopaminergic pathway that projects from the substantia nigra to the corpus striatum (Tugwell, 2008). Dauer and Przedborski (2003) propose that there are two major hypotheses for the pathogenesis of PD. One hypothesis suggests that misfolding and aggregation of proteins are key in the death of SNpc dopaminergic neurons, whereas the other hypothesis proposes that mitochondrial dysfunction and oxidative stress, including toxic oxidised DA species, are the main cause. According to Dauer and Przedborski (2003), available data argues that the mechanism of neuronal death in PD starts with a healthy dopaminergic neuron being affected by an aetiological factor such as mutant α-synuclein. On the other hand, there may be a cascade of deleterious factors within the neuron, that is made up of multiple factors such as free radicals, mitochondrial dysfunction, excitotoxicity, neuroinflammation and apoptosis (Przedborski, 2005).

2.1.1.4 Genetics

According to Lees et al. (2009), several mutations in several genes are linked with L-dopa-responsive parkinsonism. Six pathogenic mutations in LRRK-2, a kinase encoding the protein dardarin, have been reported, and the most common of these is Gly2019Ser with a world-wide frequency of 1% in sporadic cases and 4% in patients with hereditary parkinsonism (Lees et al., 2009). Lees et al. (2009) suggests that loss-of-function mutations in four genes, parkin, DJ-1, phosphatase and tensin homolog (PTEN)-induced putative kinase 1 (PINK1) and adenosine triphosphate (ATP) 13A2 cause recessive early onset parkinsonism (age of onset < 40 years). Heterozygous loss of function of GBA increases the risk of developing PD more than fivefold, thus the risk of developing PD is increased thirteen times if one carries a severe GBA mutation, which reduces mean age of disease onset from 60 to 55 years (Lees et al., 2009). Parkin mutations are the second most common genetic causes of L-dopa-responsive parkinsonism (Lees et al., 2009).

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2.1.2 Symptomatic treatment 2.1.2.1 L-Dopa

L-Dopa, the metabolic precursor of DA is the most effective medication for PD and it should always be the initial treatment option regardless of the age of the patient (Lees et al., 2009). Oral L-dopa is absorbed rapidly from the small intestines and into the CNS through an aromatic amino acid membrane transporter, and it competes with dietary protein at this level (Bruton et al., 2008). In the CNS, dopa decarboxylase converts L-dopa to DA, mainly within the presynaptic terminals of dopaminergic neurons in the striatum (Bruton et al., 2008). According to Bruton et al. (2008), L-dopa is primarily decarboxylated in the intestinal mucosa and at other peripheral sites so that little of the parent drug reaches the cerebral circulation, and probably <1% penetrates the CNS. Therefore L-dopa should not be administered alone, but together with a peripherally acting inhibitor of aromatic L-amino acid decarboxylase (AADC), that does not penetrate well into the CNS. Bruton et al. (2008) states that inhibition of peripheral decarboxylase significantly increases the fraction of administered L-dopa that remains unmetabolised and available to cross the BBB, and reduces the incidence of nausea and other gastrointestinal (GI) side-effects due to peripheral conversion of the drug to DA.

Figure 2.2: Structures of dopamine and L-dopa.

Although L-dopa is the most effective agent for symptomatic treatment of PD, it loses its efficiency with long term use, and the patient’s motor state may fluctuate severely with each dose, resulting in the so-called “on/off phenomenon” in which each dose of L-dopa effectively improves mobility for a period of time, perhaps 1 to 2 hours, but rigidity and akinesia return rapidly at the end of the dosing interval (Bruton et al., 2008).

2.1.2.2 DA agonists

DA agonists provide effective relief either as first line therapy or in early PD as an adjunct to L-dopa (Fernandez & Chen, 2007). These drugs are a common first-line treatment in patients younger than 55 years of age (Lees et al., 2009). According to Bruton et al. (2008), most DA receptor agonists have a substantially longer duration of action than L-dopa, and they are particularly effective in the treatment of patients that have developed the on/off

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phenomena. However, DA agonists are less potent than L-dopa and they do not target all symptoms of PD (Fernandez & Chen, 2007) .There are four orally administered DA agonists available for PD treatment: older ergot agents such as bromocriptine and pergolide, and two newer more selective compounds ropinirole and pramipexole (Bruton et al., 2008).

Figure 2.3 Structures of bromocriptine, ropinirole, pramipexole and pergolide.

Bromocriptine is a D2 receptor agonist and a partial D1 receptor antagonist while pergolide is an agonist of both receptor types (Bruton et al., 2008). Ropinirole and pramipexole have selective activity at D2 and D3 receptors, with little or no activity at receptors of the D1 class (Bruton et al., 2008). Bruton et al. (2008) states that initial treatment with bromocriptine or pergolide may cause nausea, fatigue and severe hypotension and should be initiated at a lower dose which can gradually be titrated over a period of weeks to months. On the other hand, ropinirole and pramipexole can be initiated more quickly, achieving therapeutically useful doses in a week or less. In addition to that, non-ergot DA receptor agonists cause lesser GI disturbances than ergot derivatives, but they can cause nausea and sleepiness (Bruton et al., 2008).

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Apomorphine

Apomorphine is a potent DA agonist which stimulates both the D1 and D2 receptors (Tugwell, 2008; Rossiter et al., 2012). It is administered subcutaneously and has high affinity for D2, D3, D5, and adrenergic α1D, α2B and α2C receptors. Apomorphine has low affinity for D1 receptors (Bruton et al., 2008). Apomorphine has a rapid onset of action and is used as “rescue therapy” for the acute treatment of the “off” response to dopaminergic therapy (Bruton et al., 2008; Tugwell, 2008; Rossiter et al., 2012). This drug has similar side effects to those of oral DA agonists. It is highly emetogenic and requires pre- and post-antiemetic therapy, starting 3 days prior to the initial dose of apomorphine, and continued at least during the first 2 months of therapy (Bruton et al., 2008).

Figure 2.4: Structure of apomorphine. 2.1.2.3 Carbidopa and benserazide

Carbidopa and benserazide are inhibitors of peripheral AADC that do not penetrate well into the CNS. AADC is an enzyme that decarboxylates L-dopa, thus decreasing its conversion to DA in the peripheral tissues, and resulting in a decrease in its CNS bioavailability (Bruton et al., 2008). Inhibition of this enzyme markedly increases the fraction of administered L-dopa that remains unmetabolized and available to cross the BBB (Dhall & Kreitzman, 2016). Formulations of L-dopa with AADC inhibitors allows administration of lower doses of L-dopa, thus reducing peripheral side-effects (Carvey, 2010).

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2.1.2.4 COMT inhibitors

Catechol-O-methyltransferase (COMT) metabolises L-dopa as well as DA, resulting in the pharmacologically inactive compounds O-methyl dopa (from L-dopa) and 3-methoxytyramine (from DA) (Bruton et al., 2008). COMT inhibitors block this peripheral conversion of L-dopa to 3-O-methyl dopa, increasing both plasma half-life (t1/2) of L-dopa as well as the fraction that reaches the CNS (Bruton et al., 2008). COMT inhibitors can be used adjunctively with L-dopa in late phases of PD, reducing both the “off” time experience, and the end-of-dose deterioration (Tugwell, 2008; Rossiter et al., 2012).

There are two COMT inhibitors currently available for the treatment of PD namely entacapone and tolcapone. Entacapone increases bioavailability of L-dopa by inhibiting COMT peripherally and does not cross the BBB (Tugwell, 2008). Several studies have shown that entacapone reduces motor fluctuations in patients with PD. Tugwell (2008) adds that there is a possibility that co-administration of entacapone with L-dopa may delay the development of motor fluctuations.

Figure 2.6: Structures of tolcapone and entacapone 2.1.2.5 MAO-B inhibitors

MAO-B inhibitors block the B isoform of the MAO enzyme that is found in the human brain (Fernandez & Chen, 2007). Selectively inhibiting this enzyme enhances striatal dopaminergic activity by inhibiting the oxidative metabolism of DA, thus improving the motor symptoms of PD (Fernandez & Chen, 2007). Two drugs, selegiline [(R)-deprenyl] and rasagiline, are currently the most used inhibitors of MAO-B (Tugwell, 2008). Selegiline is used as an adjunct to L-dopa in the management of PD, whereas rasagiline is used as mono- or adjunct therapy (Rossiter et al., 2012).

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Figure 2.7: Structures of selegiline and rasagiline 2.1.2.6 Anticholinergic drugs

Before the discovery of L-dopa, antagonists of muscarinic ACh receptors were widely used for treatment of PD (Bruton et al., 2008). Anticholinergics were first used when it was discovered that dopaminergic deficiency resulted in increased striatal cholinergic activity and a subsequent imbalance between these neurotransmitters (DeMaagd & Philip, 2015). Anticholinergic drugs can be used as adjuncts to L-dopa as well as monotherapy early in the course of the disease (Brocks, 1999).

Bruton et al. (2008) suggests that anticholinergic drugs act in the neostriatum through the receptors that mediate the response to intrinsic cholinergic innervation. The agents acting as muscarinic antagonists that are currently used in treatment of PD include trihexyphenidyl, benztropine and diphenhydramine (Bruton et al., 2008). Trihexyphenidyl is a synthetic tertiary amine anticholinergic that is used as an adjunct to L-dopa therapy. This drug decreases striatal levels of Ach without increasing its turnover rate (Meltzer, 1991). Benztropine acts with a similar mechanism to trihexyphenidyl (Meltzer, 1991).

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Figure 2.8: Structures of trihexyphenidyl, diphenhydramine, and benztropine. 2.1.2.7 Adenosine A2A receptor antagonists

Hauser and Schwarzschild (2005) state that specific adenosine A2A receptor antagonists reverse motor deficits or enhance dopaminergic treatments in animal models of PD. A2A blockade has been found to improve abnormalities of muscle tone and tremors in rodents, extending the potential benefits of A2A blockade for PD symptoms. Hauser and Schwarzschild (2005) state that co-administration of an A2A receptor antagonist with L-dopa produces a synergistic antiparkinsonian effect. A selective adenosine A2A antagonist istradefylline remediates PD by blocking A2A receptor-mediated striatopallidal medium spiny neuron modulation, thus facilitating nigral neurotransmitter release (Dungo & Deeks, 2013). A2A antagonists also have the potential for improving motor activity while showing a low potential for inducing or exacerbating dyskinesia. In addition, laboratory studies have raised the possibility that prolonged A2A blockade may prevent the development of dyskinesia (Hauser & Schwarzschild, 2005).

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Figure 2.9: Structure of istradefylline (KW-6002) 2.1.2.8 Amantadine

Amantadine was originally used as an antiviral drug and its antiparkinson effects were discovered in the late 1990s (Tugwell, 2008). Amantadine has anticholinergic properties, but more importantly, it can activate DA release from nerve terminals (Fahn, 2003; Bruton et al., 2008). In addition, amantadine interferes with transmission at glutamatergic N-methyl-D-aspartate (NMDA) receptors, and in turn inhibits NMDA-evoked release of ACh in striatal tissue (Tugwell, 2008). It has been suggested that amantadine may be useful as an adjunct to L-dopa. Its glutamate-antagonist properties may reduce overactivity of the subthalamic nucleus which may be the cause of dyskinesia, thus reducing the severity of L-dopa-induced dyskinesia (Fahn, 2003; Bruton et al., 2008; Tugwell, 2008).

Figure 2.10: Structure of amantadine 2.1.3 Drugs for neuroprotection

2.1.3.1 MAO-B inhibitors: Selegiline, lazabemide and L-dopa

The tocopherol and deprenyl antioxidative therapy of parkinsonism (DATATOP) study conducted with selegiline can delay the emergence of disability that require treatment with L-dopa (Fernandez-Espejo, 2004). In contrast to L-L-dopa monotherapy, co-administration of selegiline with L-dopa prolongs chances of survival and lessens disability (Koller, 1997). Clinical trials have led to a hypothesis that chronic selegiline would lessen oxidative stress generated from DA turnover, and afford neuroprotection (Koller, 1997; LeWitt & Taylor, 2008).

Studies have demonstrated that selegiline limits MPTP-induced nigral damage and reduces PC12 cell apoptosis in doses that do not inhibit MAO-B (Koller, 1997). It has been proposed that selegiline rescues nigral neurons by inducing selective changes in transcription, protein synthesis and alterations in gene expression. Research has also shown that selegiline, in

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doses that do not inhibit MAO-B, limits free radical formation and prevents nigral damage due to direct administration of MPP+_{(Koller, 1997). Selegiline may influence the rate of} neuronal degeneration through mechanisms that are not dependent on MAO-B inhibition (Koller, 1997). In addition, treatment with selegiline lowers the risk for eventually developing freezing of gait (Koller, 1997).

Figure 2.11: Structure of lazabemide.

Lazabemide is a selective reversible MAO-B inhibitor and undergoes rapid clearance after administration. It has been demonstrated that lazabemide has similar symptomatic effects compared to selegiline (LeWitt & Taylor, 2008). Rasagiline, a highly selective MAO-B inhibitor, enhances release of DA in addition to retarding its catabolism and antagonises three cellular processes that are involved in the cascade of events leading to apoptosis. These events are intracellular translocation of glycolytic enzyme glyceraldehyde-3-phosphate, induction of bcl-2 and activation of mitochondrial permeability transition (LeWitt & Taylor, 2008). Rasagiline also blocks actions of N-methyl-R-salsolinol, an endogenous neurotoxin that is a condensation product of oxidised DA (LeWitt & Taylor, 2008).

2.1.3.2 Dopaminergic drugs: Pramipexole, ropinirole and rasagiline

Studies have shown that initial treatment of PD patients with DA agonists, pramipexole or ropinirole, reduces the decline of nigrostriatal function compared to patients on L-dopa alone (Fernandez-Espejo, 2004). DA agonists such as pramipexole have been demonstrated to exert a direct antioxidant effect and they scavenge hydroxyl radicals as a result of their hydroxylated benzyl ring structure, thus rendering them neuroprotective (Fernandez-Espejo, 2004). DA receptor agonists have been hypothesised as potentially neuroprotective by acting at D2 autoreceptors, found in dopaminergic substantia nigra terminals, to supress DA release and thus reduce oxidative stress (Yacoubian & Standaert, 2009). According to Yacoubian and Standaert (2009) in vitro and animal studies have shown that DA receptor agonists reduce dopaminergic cell death.

Studies with pramipexole have demonstrated potentially protective actions against oxidative stress and the neurotoxic effect on dopaminergic neurons of various experimental toxins including methamphetamine, 3-acetylpyridine, 6-OHDA and MPTP (LeWitt & Taylor, 2008). However, the mechanisms contributing to the protective actions of pramipexole have not

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been defined, although activation of the D3 receptor agonist was suggested in one study, and blocking the cascade of apoptosis in others (LeWitt & Taylor, 2008).

Ropinirole was shown to enhance mechanisms against oxidative stress and exerts protective action against 6-OHDA-induced loss of nigrostriatal dopaminergic projections in mice (LeWitt & Taylor, 2008).

2.1.3.3 Antioxidant therapy

Studies have shown that selegiline significantly delays the time of onset of L-dopa treatment (Yacoubian & Standaert, 2009). On the other hand, rasagiline is more potent than selegiline and has metabolites with potential antioxidant properties. The antioxidant, coenzyme Q10, is a cofactor in the electron transport chain in mitochondria and it has been shown to reduce dopaminergic neurodegeneration in PD mouse models (Yacoubian & Standaert, 2009). Yacoubian and Standaert (2009) state that uric acid is a potential neuroprotective agent in PD. Uric acid acts as an antioxidant by scavenging reactive oxygen and nitrogen species. A decreased incidence of PD among patients with high serum urate levels and among subjects with gout has been reported in previous studies (Yacoubian & Standaert, 2009). Slower disease progression is attributed to high plasma urate levels in patients with early PD (Yacoubian & Standaert, 2009).

2.1.3.4 Mitochondrial energy enhancement drugs: Coenzyme Q10 and creatine

Figure 2.12: Structures of coenzyme Q10 and creatine

Mitochondria of the substantia nigra, platelets and skeletal muscle in PD possess reduced activity of the first step of the mitochondrial electron transport chain, complex I (LeWitt & Taylor, 2008). Coenzyme Q10 (also known as ubiquinone) is an essential co-factor serving as an electron acceptor for mitochondrial complex I (LeWitt & Taylor, 2008). Several studies have shown that coenzyme Q10 is also a potent antioxidant in lipid membranes and mitochondria as it has a potential of slowing PD progression. LeWitt and Taylor (2008) state that augmenting brain creatine concentration is another strategy for targeting the defects in mitochondrial complex I.

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According to LeWitt and Taylor (2008), creatine serves as a precursor for conversion to phosphocreatine, an energy intermediate which transfers phosphoryl groups for ATP synthesis in mitochondria. Increasing creatine intake enhances formation of phosphocreatine, ultimately resulting in the reduction of oxidative stress though stabilisation of mitochondrial creatine kinase (LeWitt & Taylor, 2008). In addition, creatine kinase inhibits the opening of the mitochondrial transition pore, hence improving mitochondrial metabolism and down regulating a putative neurodegenerative mechanism (LeWitt & Taylor, 2008).

2.1.3.5 Anti-inflammatory drugs

Activation of microglia, increased cytokine production, and increased complement protein levels suggestive of neuroinflammation have been demonstrated in PD (Yacoubian & Standaert, 2009). According to Yacoubian and Standaert (2009), anti-inflammatory agents, including non-steroidal anti-inflammatory drugs (NSAIDs) and minocycline, have been pursued as potential disease modifying treatments for PD in order to slow down disease progression. Certain NSAIDs, such as aspirin, have shown neuroprotective properties in studies done in culture and animal models (Yacoubian & Standaert, 2009). However, there is conflicting data regarding which NSAID, what dosing and what timing provides the best neuroprotection (Yacoubian & Standaert, 2009). Research has indicated that the use of NSAIDs lowers the risk of PD by 45%, but ibuprofen is the only NSAID with proven neuroprotective effect (Yacoubian & Standaert, 2009).

2.1.3.6 Antiapoptotic drugs: Minocycline, TCH346 and Cep-1347

There is evidence that activation of apoptosis is a possible mechanism for neurodegeneration in PD. Studies show that pre-treatment with minocycline improves survival of dopaminergic substantia nigra neurons in rodent models of 6-OHDA and MPTP-induced parkinsonism (LeWitt & Taylor, 2008). According to LeWitt and Taylor (2008), minocycline inhibits the activation of microglia, which is a prominent feature in the brain of PD patients and in experimental neurotoxin models. Minocycline also lessens factors that mediate apoptosis, such as caspase-I (LeWitt & Taylor, 2008).

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), an enzyme that can initiate apoptosis, is inhibited by the propagylamine TCH346, which has antiapoptotic activity (Yacoubian & Standaert, 2009). TCH346 has a similar structure to selegiline, however, it does not inhibit MAO-B and it is not metabolised to amphetamine compounds (LeWitt & Taylor, 2008). Histological analysis has revealed that TCH346 treatment lessens MPTP induced dopaminergic neuron loss (LeWitt & Taylor, 2008).

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Another antiapoptotic agent that has shown promise in clinical studies is CEP-1347 (Yacoubian & Standaert, 2009). This compound is an inhibitor of mixed lineage kinase-3, a kinase that can activate the c-Jun N-terminal kinase (JNK) pathway involved in cell death (LeWitt & Taylor, 2008; Yacoubian & Standaert, 2009).

Figure 2.13: Structures of minocycline, TCH346 and Cep-1347 2.1.3.7 NMDA antagonists/ Antiglutamatergic drugs

One rationale for PD neuroprotection has been to block glutamate neurotransmission in the substantia nigra because glutamate can act as an excitotoxin contributing to neuronal damage (Tugwell, 2008). Amantadine has been claimed to slow progression of PD by reducing the extent of cell death caused by excess glutamate activity. However, there is no good evidence to support this claim (Tugwell, 2008). On the other hand, riluzole, a drug used to treat amyotropic lateral sclerosis, blocks the presynaptic release of glutamate (LeWitt & Taylor, 2008; Tugwell, 2008). However, the results of a trial with riluzole in early PD showed no evidence for a neuroprotective effect (LeWitt & Taylor, 2008; Tugwell, 2008).

2.1.3.8 Adenosine A2A receptor antagonists

Epidemiological studies have indicated that caffeine, an adenosine A2A antagonist, may reduce the incidence of PD at least in men (Hauser & Schwarzschild, 2005). Caffeine mediates its action by antagonising adenosine receptors when administered to mice in

Design, synthesis and evaluation of quinazolinone analogues as monoamine oxidase inhibitors